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Purification and Analysis of Rho‐1D4‐tagged Peripheral Cannabinoid Receptor CB2
Author(s) -
LocatelliHoops Silvia Cristina,
Gorshkova Inna,
Gawrisch Klaus,
Yeliseev Alexei A
Publication year - 2013
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.27.1_supplement.790.18
Subject(s) - chemistry , monoclonal antibody , recombinant dna , fusion protein , affinity chromatography , maltose binding protein , receptor , surface plasmon resonance , epitope , rhodopsin , ligand (biochemistry) , microbiology and biotechnology , biochemistry , antibody , chromatography , biology , enzyme , nanoparticle , retinal , immunology , gene , materials science , nanotechnology
Human peripheral cannabinoid receptor CB 2 is involved in regulation of the immune response. For studies requiring surface immobilization, pure and functional receptor with a reliable tag is required. The goal of this project is to test the usefulness of the C‐terminal epitope of rhodopsin (rho‐1D4‐tag) for purification and surface immobilization of recombinant CB 2 using the monoclonal antibody 1D4. E xpression of rho‐1D4‐tagged CB 2 as a fusion with maltose binding protein (MBP) in E coli was evaluated in terms of protein levels, accessibility of the rho‐1D4 tag and activity. Highly pure tagged CB 2 was obtained by rho‐1D4 and Ni‐NTA affinity chromatography. The reconstituted protein was functionally active by G protein activation assay. The interaction of rho‐1D4‐tagged CB 2 with 1D4 antibody was characterized by surface plasmon resonance (SPR). Either purified or fusion CB 2 from crude extracts was captured on the 1D4 antibody‐coated surface in a specific and quantitative fashion, and its presence confirmed by biding of an anti‐CB 2 monoclonal antibody. Ligand binding to purified receptor in detergents was studied upon immobilization of fusion CB 2 on 1D4 antibody resin. This methodology was also used to study thermal stability of the receptor in micelles. In conclusion, the rho‐1D4 tag was successfully utilized for purification, immobilization, biochemical and biophysical studies of functional CB 2 .